Biocontrol of plant diseases caused by Fusarium species with novel isolates of Pantoea agglomerans

ABSTRACT

Microbiological agents are provided for control of certain diseases of wheat and other cereals caused by Fusarium species, including Fusarium head blight of wheat and other cereals. These agents can also improve yield of wheat plants and cereals. The agents are novel isolates of  Pantoea agglomerans  and of  Bacillus megaterium  that exhibit the property of inhibiting fungal pathogens, particularly those produced by Fusarium species.  
     Biocontrol compositions, and methods of using them to control plant pathogen development on wheat and cereal plants and for increasing plant yield, are also provided. The biocontrol compositions comprise a mixture of at least one microorganism selected from the group consisting of  Pantoea agglomerans  and  Bacillus megaterium.

FIELD OF THE INVENTION

[0001] The present invention is related to the biological control ofplant diseases caused by Fusarium species. Specially, it relates tobiocontrol compositions comprising a mixture of at least onemicroorganism which is an antagonist against plant pathogens and anappropriate carrier, as well as to a process for control of the plantpathogen and to increase yield. The invention includes as a plantpathogen the pathogenic fungus Gibberella zeae (anamorph Fusariumgraminearum), and as antagonist microorganisms the novel isolates ofPantoea agglomerans (Embr. 1494, Accession ATCC PTA 3460) and ofBacillus megaterium (Embr. 9790, Accession ATCC PTA 3461).

BACKGROUND OF INVENTION

[0002] Fruit, vegetables, and plants are all susceptible to attack byfungi, resulting in loss of crops, decreased shelf-life of produce, andultimately higher costs for consumers. Many fungi are known pathogens inseveral diseases which harm or destroy crops worldwide. Examples of suchfungi include those belonging to the genera Rhizoctonia, Pythium,Gaeumannomyces, and Fusarium.

[0003] For a number of years, it has been known that variousmicroorganisms exhibit biological activity useful in controlling plantdiseases. Although progress has been made in the field of identifyingand developing biological pesticides for controlling plant diseases ofagronomic and horticultural importance, most of the pesticides in useare still synthetic compounds. Many of the chemical fungicides arecarcinogenic agents and, therefore, toxic to wildlife and othernon-target species. In addition, pathogens may develop resistance tochemical pesticides. In fact, the fungicides, considered the majorweapon in combating plant diseases, are often ineffective and posehazards to humans and the environment. Biological control offers anattractive approach as compared with synthetic chemical fungicides.Biopesticides (living organisms and the compounds which are naturallyproduced by these organisms) can be safer, more biodegradable, and lessexpensive to develop. In addition, they are highly desired forintegrated pest management programs in agriculture, public health, andurban settings.

[0004] The agricultural use of Bacillus megaterium has been reported fordisease control in rice and cotton inhibition as seed treatment but notas foliar sprays. U.S. Pat. No. 5,403,583 discloses a Bacillusmegaterium, ATCC 55000, and a method to control the fungal plantpathogen Rhizoctonia solani as seed treatment. Islam and Nandi alsodisclosed a Bacillus megaterium with antagonism to Drechslera oryzae,the causal agent of rice brown spot (Journal of Plant Diseases andProtection. 92(3): 241-246 (1985) and a Bacillus megaterium with invitro antagonism against Drechslera oryzae, Alternaria alternata andFusariun roseum (Journal of Plant Diseases and Protection. 92(3):233-240 (1985). They mentioned three components in the culture filtrate.The most active antibiotic was highly soluble in water and methanol witha UV peak at 255 nm and a shoulder at 260 nm, which proved to be apolyoxin-like lipopeptide. And, Cook (Proceedings Beltwide CottonProduction-Mechanization Research Conference, Cotton Council, Memphis,p. 43-45 (1987) disclosed the use of a suspension of Bacillus megateriumto reduce the number of plants killed by Phymatotrichum omnivorum, acause of cotton root rot. Antibiotic production of B. megaterium hasalso been recorded by Berdy (CRC Handbook of Antibiotic Compounds, Vols.I-XIV. CRC Press, Inc. Boca Raton. Fla. 1980-87), who reported theproduction of low-mammalian toxic peptide antibiotics such asansamitocin-PDM-O, bacimethrin, megacin, pentapeptide, and homopeptides.

[0005] U.S. Pat. No. 5,494,819 describes a pure culture of Pantoeaagglomerans having all of the characteristics of FERM BP-3511 which isidentified by growth, morphology, physiology, utilization of carbonsources and various specific enzymatic tests involving enzymes as lysinedecarboxylase, arginine dihydroxylase, phenylalanine deaminase andornithine decarboxylase. In addition, the disclosed pure culture ofPantoea agglomerans is characterized by the required production oflipopolysaccharides to which the inventors attribute animmunity-stimulating activity. In other words, according to thisdocument, Pantoea agglomerans is used to obtain substances to be used inpharmaceuticals.

[0006] U.S. Pat. No. 5,766,926 discloses a method comprising the stepsof applying to the pulpwood or pulp substrate a bacterial inoculum of atleast one of the species selected from the group consisting ofPseudomonas fluorescens, Pantoea (Enterobacter) agglomerans, Bacilluscereus, and Xanthomonas campestris and maintaining the substrate underconditions which allow bacterial growth for a time sufficient to effecta reduction in the resin component of the substrate by the bacteria. Itis mentioned that the source of the Pantoea (Enterobacter) agglomeransisolate used in the method, identified by the NRRL Accession No.B21509,is Brazil.

[0007] It is known that the genus Fusarium contains species which maycause diseases of wither and blight that occur during the growth ofplants and damages not only the host but also other kinds of plants. Itis supposed that fusaric acid is the principal agent that brings aboutthese diseases. Fusaric acid (5-n-butylpicolinic acid) is known to be anon-specific toxin which is produced by the metabolism of almost allplant pathogenic Fusarium fungi (Wood, R. K. et al. 1972. “Phytotoxinsin plant diseases”. Academic Press. New York; Durbin, R. D. 1982.“Toxins in plant diseases”. Academic Press. New York). In the documentEP 257 756, referring to the prevention of Fusarium diseases andmicroorganisms therefor, the inventors proposed to prevent such diseasesby using microorganisms belonging to the genera Cladosporium andPseudomonas which decompose fusaric acid. EP 441 520 relates also todetoxifying fusaric acid microorganisms, and Klebsiella oxytoca HY-1(FERM BP-3221) is particularly mentioned.

[0008] In the document WO 92018613, it is suggested to control plantdiseases caused by fungi of the genera Rhizoctonia, Pythium, andFusarium by using a new strain of Pseudomonas fluorenscens, a seed orsoil treatment but not foliar sprays.

[0009] WO 9905257, referring to biocontrol for plants with Paenibacillusmacerans, Pseudomonas putida, and Sporobolomyces roseus, describes theuse of isolates of these microorganisms to impart pathogen protection toplants, particularly against plant diseases caused by fungi, such asFusarium oxysporum, Fusarium graminearum, Fusarium monilforme,Cochliobolus sativus, Collectotrichum graminicola, Stagonospora nodorum,Stagonospora avenae, Stenocarppela maydis, and Pyrenophoratritici-repentis. In this case, pathogen protection was achieved byeither seed treatment or foliar sprays.

[0010]Fusarium graminearum Schw. (Teleomorph=Gibberella zeae Schw.Petch.) is the Fusarium species most frequently responsible for scab ofwheat and barley in Brazil. This disease, also known as Fusarium HeadBlight (FHB), is responsible for major losses which vary from 10% (seeLuz, W. C. da. 1984. “Yield losses caused by fungal foliar wheatpathogens in Brazil”. Phytopathology. 74:1403-1407); to 54% (Picinini,E. C. and Fernandes, J. M. C. 1994. “Controle quimico da Gibberella zeaeem trigo pelo uso de fungicidas inibidores da sintese do ergoterol”.Fitopatol. Brasileira 19 (Supl.):273). At present, available andaffordable control measures, such as resistant varieties, culturalpractices, and foliar fungicides, are only partially effective.

[0011] Only modest levels of resistance have been deployed in cultivarsin commercial fields; the most widely grown cultivars are often mostsusceptible. Furthermore, the benefit of crop rotation as a controlmeasure is reduced by the wide host range of the pathogen, especially ongrasses (Costa Neto, J. P. da. 1976. “Lista de fungos sobre gramineas(capins e cereais) no Rio Grande do Sul”. Revista da Faculdade deAgronomia. UFRGS. 1:43-78; Luz, W. C. da. 1982. “Diagnose das Doencas daCevada”. Passo Fundo—EMBRAPA-CNPT, 24p. (Circular Técnica no. 2)).Treatment with foliar fungicides remains the most important (Picininiand Fernandes, 1994) and recommended (Reunião da Comissão Sul-Brasileirade Pesquisa de Trigo, 2000) tool for reducing scab in Brazil, despiteits shortcomings as a control measure. The use of certain effectivefungicides has been restricted in some countries because application atlate developmental stages, that is, during heading and flowering, canresult in chemical residues in the harvested grain. Biological controlis an additional strategy that may eventually play an important role inan integrative approach to scab management of cereals.

[0012] Screening of microorganisms to control wheat scab was initiatedin Brazil in the 1980's (Luz, W. C. da. 1988. “Biocontrol of fungalpathogens of wheat with bacteria and yeasts”. Page 348 in: 5^(th)International Congesss of Plant Pathology, Kyoto, Japan. (Abstr.)). Atthe beginning, over 300 bacteria and yeasts isolated from wheat werescreened in vitro against F. graminearum. This work was followed by thatof Perondi et al. (Perondi, N. L., Thomas, R. and Luz, W. C. da. 1990.“Antagonistas potenciais de Fusarium graminearum”. In: Anais do 2°Simpósio de Controle Biológico, Brasilia, D. F., p. 128. (Abstr.);Perondi, N. L., Thomas, R. and Luz, W. C. da. 1990. “Controle microbianoda giberela do trigo em campo”. In: Anais do II Simpósio de ControleBiológico, Brasilia, D F. P.129(Abstr.); Perondi, N. L., Luz, W. C. da.and Thomas, R. 1996. “Controle microbiológico da giberela do trigo”.Fitopatol. Brasileira 21:243-249) in which microbial strains were testedfor their antagonistic action against the pathogen. Potentialantagonists were selected by the funnel method (Luz, W. C. da. 1990.“Microbiological control of Bipolaris sorokiniana ‘in vitro’”.Fitopatol. Brasileira 15:246-247) which compared the effect ofindividual test organisms on the radial growth of F. graminearum.Promising isolates were further tested in the greenhouse and in thefield for their ability to control wheat scab. Individual bioprotectantssignificantly diminished the severity of the disease under fieldconditions, raising the yield of wheat between 7 and 31% when comparedto non-treated plants.

[0013] Besides the selection of the bioprotectants, it is important toovercome several difficulties related to constraints on theirapplication to the ears of wheat and barley at flowering such as thetiming of application, inoculation technology, physiological state ofthe organisms, spike colonization, survival of the organisms under theharsh environmental conditions, variability of biocontrol from year toyear, fermentation, formulation, and storage. The partial control of anytactics to protect against FHB up to this moment indicates that theintegration of protection measures would provide the best diseasemanagement.

[0014] From 1988 up to now, thousands of microorganisms have been testedfor scab control. Some workers have been investigating antagonists tocontrol FHB (Khan, N. J., Schisler, D. A., Boehm, M. J., Lipps, P. E.,Slininger, P. J. and Bothast, R. J. 1998. “Biological control of scab ofwheat incited by Giberella zeae”. Pages 45-46 in: Proceedings of the1998 National Fusarium Head Blight Forum, Michigan State University,University Printing, East Lansing. Mich.; Khan, N. J., Schisler, D. A.,and Boehm, M. J. 1999. USDA-ARS, Ohio State University cooperativeresearch on biologically controlling Fusarium Head Blight: 2. Influenceof pathogen strain, inoculum spray sequence and inoculum spray time.Pages 56-59 in: Proceedings of the 1999 National Fusarium Head BlightForum, Michigan State University, University Printing, East Lansing,Mich.; Boehm, m. J., Khan, N. J., and Schisler, D. A. 1999. USDA-ARS,Ohio State University cooperative research on biologically controllingFusarium Head Blight: 3. Field testing of antagonists. Pages 45-48 in:Preceedings of the 1999 National Fusarium Head Blight Forum, MichiganState University, University Printing, East Lansing, Mich.; Luo, Y. andBleakley, B. 1999. “Biological control of Fusarium Head Blight (FHB) ofwheat by Bacillus strains”. Pages 78-81 in: Proceedings of the 1999National Fusarium Head Blight Forum, Michigan State University,University Printing, East Lansing, Mich.; Schisler, D. A., Khan, N. J.,and Boehm, M. J. 1999. USDA_ARS, Ohio State University cooperativeresearch on biologically controlling Fusarium Head Blight: 1. Antagosistselection and testing on durum wheat. Pages 78-81 in: Proceedings of the1999 National Fusarium Hrad Blight Forum, Michigan State University,University Printing, East Lansing, Mich.; Stockwell, C. A., Luz, W.C.da., and Bergstrom, G. C. 1997. “Biocontrol of wheat scab withmicrobial antagonists”. Phytopathology 87:S94.(Abstr.); Stockwell, C.A., Bergstrom, G. C., and Luz, W. C.da.1999. “Selection of microbialantagonists for biological control of Fusarium Head blight of wheat”.Pages 82-84. in: Proceedings of the 1999 National Fusarium Head BlightForum, Michigan State University, University Printing, East Lansing,Mich.; Stockwell, C. A., Bergstrom, G. C., and Luz, W. C.da.2000.“Identification of bioprotectants for biological control of Gibberellazeae” in:. Proceedings of FHB Forum), under greenhouse or fieldconditions. Some strains have reduced the FHB severity and significantlyreduced vomitoxin contamination in grains (Stockwell et al., 1997,2000).Table 1 illustrates the chronology of researches on the biocontrol ofFHB. TABLE 1 Chronology of works done on biocontrol of Fusarium HeadBlight of wheat LITERATURE BIOPROTECTANTS Lus, W.C. da, 1988 Bacteria,Yeast Perondi; N.L., Luz, W.C. da & Thomas, R, Bacillus subtilis 1990 a,1990 b, 1996 Bacillus spp. Pseudomonas fluorescens Sporobolomyces roseusStockwell, C.A; Luz, W.C. da, and Paenibacillus macerans Bergstrom, G.C.1997 Pseudomonas putida Sporobolomyces roseus Khan, N.I., Scisler, D.A..Bochm, M.J, Bacillus spp. Lipps, P.E., Slininger, P.J. and Bothast,R.J., 1998 Boehm, M.J., Khan., N.J., and Schisler, Yeast, Bacillus sp.D.A, 1999 Khan, N.J., and Schisler, D.A., and Yeast, Bacillus sp. Boehm,M.J., 1999 Luo, Y. & Bleakely, B. 1999 Bacillus spp. Schisler, D.A.,Khan, N.J. and Boehm, Bacillus spp. M.J. 1999 Stockwell, C.A.,Bergbstrom, G.C. and Paenibacillus macerans Luz, W.C. da. 1999Pseudomonas putida Sporobolomyces roseus Stockwell, C.A., Bergstrom,G.C. and Paenibacillus macerans Luz, W.C. da., 2000 Bacillus spp.

SUMMARY OF THE INVENTION

[0015] According to the present invention, microbiological agents areprovided for control of certain diseases of wheat and other cerealscaused by Fusarium species, including Fusarium Head Blight (FHB) ofwheat and other cereals. Moreover, these agents can also improve yieldof said wheat plants and cereals. Specifically, these agents are novelisolates of Pantoea agglomerans and of Bacillus megaterium that exhibitthe property of inhibiting fungal pathogens, particularly those producedby Fusarium species.

[0016] The first embodiment of the invention refers to a biocontrolcomposition comprising a mixture of at least one microorganism which isantagonist against plant pathogens and a carrier for said at least onemicroorganism, wherein said at least one microorganism is a bacteriaselected from the group consisting of Pantoea agglomerans and Bacillusmegaterium and said at least one microorganism is present in an amounteffective for inhibiting plant pathogen development.

[0017] A second embodiment is related to a process for controlling theplant pathogen development on wheat and cereal plants by applying acomposition containing a carrier and at least one microorganism which isan antagonist against plant pathogens selected from the group ofbacteria consisting of Pantoea agglomerans and Bacillus megaterium in anamount effective to inhibit plant pathogen development on said plant.

[0018] The third embodiment is related with a process for increasingplant yield characterized by a step of applying, particularly byspraying, to the plant a composition containing a carrier and at leastone microorganism selected from the group of bacteria consisting ofPantoea agglomerans and Bacillus megaterium in an amount effective toincrease yield of said plants or plants resulting from treated seeds.

DETAILED DESCRIPTION OF THE INVENTION

[0019] For purposes of clarity and a complete understanding of theinvention, the following terms are defined.

[0020] “Plants” is used to mean the head part of the plant to betreated.

[0021] “Pantoea agglomerans (Embr. 1494)” means the bacterium isolatewhich was isolated by Embrapa and identified by the code “Embr. 1494”.

[0022] “Bacillus megaterium (Embr. 9790)” means the bacterium isolatewhich was isolated by Embrapa and identified by the code “Embr. 9790”.

[0023] “CFU” refers to the abbreviation of Colony Forming Unity which isfrequently used to express the concentration of microorganisms presentin a composition.

[0024] Microorganisms usable in the present invention were identified bythe following procedure: (i) screening plants or agriculturalcommodities (e.g. the surface(s) of said plant or agriculturalcommodity) for the presence of useful microorganisms; (ii) recovering(e.g. by washing or rising from the plant or agricultural commodity) andisolating said microorganism(s); and (iii) testing said microorganism(s)for antagonistic activity against plant pathogens. However, it should beunderstood that said microorganism(s) may be obtained from sources otherthan said plants or agricultural commodities.

[0025] The isolates of the present invention, Accession No. ATCC PTA3460 (Embr. 1494) and Accession No. ATCC PTA 3461 (Embr. 9790), wereobtained from wheat or corn plant parts, such as healthy leaves, seedsor roots by repeatedly washing the plant parts with water. The organismswere thereafter plated and grown on any nutritionally rich mediumsufficient to support growth of the organisms. Preferably, the medium isnutrient agar. ATCC PTA 3460 was identified as a novel isolate ofPantoea agglomerans (Embr. 1494) and ATCC PTA 3461 was identified as anovel isolate of Bacillus megaterium (Embr. 9790).

[0026] Isolate ATCC PTA 3461 (Embr. 9790) of B. megaterium has thefollowing characteristics: it is a Gram-positive rod, spore-formingbacteria, and the bacterial identification was accomplished based on 16SrRNA gene sequence similarity (made by Microbe Inotech Laboratories, Incon Apr. 21, 2000 by using PE Applied Biosystem's MicroSeq™ microbialidentification software and database) demonstrating that the isolate isnovel and belongs to the species Bacillus megaterium (details about thischaracterization method may be found in Stackebrandt, E. and Goebel, B.M. 1994. “Taxonomic Note: A Place for DNA-DNA Reassociation”; and 16SrRNA Sequence Analysis in the Present Species Definition inBacteriology. Int. J. Syst. Bacteriol. 44:846-849).

[0027] Isolate ATCC PTA 3460 (Embr. 1494) of P. agglomerans has thefollowing characteristics: it is a Gram-negative bacteria; and, based onfat acid analysis (CG FAME method), has a similarity coefficient of0.648 and distance coefficient of 3.310 (a good match is one with asimilarity coefficient greater than 0.5 and a distance coefficient ofless than 7) made by the same laboratory.

[0028] Growth of isolates ATCC PTA 3460 (Embr. 1494) and ATCC PTA 3461(Embr. 9790) may be effected under aerobic condition at any temperaturesatisfactory for growth of the microorganisms, i.e., from about 10° C.to about 30° C. The preferred temperature range is 20° C. to 25° C. ThepH of the nutrient medium is about neutral, i.e., 6.6 to 7.3. Theincubation time is that time necessary for the isolates to reach astationary phase of growth, preferably between 40 and 60 hours. Growthof isolates ATCC PTA 3461 (Embr. 9790) (B. megaterium) and ATCC PTA 3460(P. agglomerans) (Embr. 1494) is preferably achieved at a temperaturerange of 21° C. to 23° C., with an incubation time of 45 to 50 hours,such that the cells are in the logarithmic phase of growth.

[0029] Isolates ATCC PTA 3461 (Embr. 9790) (B. megaterium) and ATCC PTA3460 (Embr. 1494) (P. agglomerans) may be grown in any conventional testtube or shake flask for small fermentation runs. For large scaleoperations, the culture may be carried out in a suitable fermentationtank, under appropriate conditions provided by agitating and aeratingthe inoculated liquid medium. Following incubation, the isolates areharvested by conventional sedimentary methods (e.g. centrifugation) orfiltering. Cultures are stored on nutrient agar at 4° C., but also atmuch lower temperature such as −170° C.

[0030] The bacteria of the present invention are useful to control plantpathogens by using, for example, air spraying.

[0031] The microorganisms of the present invention may be applied towheat plants or other cereals in combination with various liquid and/orsolid carriers and additives, including combination with fungicides.

[0032] In the liquid form, e.g. solutions or suspensions, themicroorganisms may be mixed or suspended in water or in aqueoussolutions. Suitable liquid diluents or carriers include water, aqueoussolutions, petroleum distillates, or other liquid carriers.

[0033] Solid compositions can be prepared by dispersing the antagonistmicroorganisms in and on an appropriately divided solid carrier, such aspeat, wheat, bran, vermiculite, clay, talc, bentonite, diatomaceousearth, fuller's earth, pasteurized soil, and the like. When suchformulations are used as wettable powders, biologically compatibledispersing agents such as non-ionic, anionic, amphoteric, or cationicdispersing and emulsifying agents can be used.

[0034] In a preferred embodiment, the compositions contemplated hereinprevent attack by Fusarium diseases upon plants, particularly cerealplants, such as wheat, barley, and corn and, when used in sufficientamounts, to act as fungi antagonist. They have a high margin of safetybecause they do not burn or injury the plant.

[0035] The compositions of the invention are so chemically inert thatthey are compatible with substantially any other constituents of thespray schedule. They may also be used in combination with biologicallycompatible pesticidal active agents as for example, herbicides,nematocides, fungicides, insecticides, and the like. They can also beused in combination with plant growth affecting substances, such asfertilizers, plant growth regulators, and the like, provided that suchcompounds or substances are biologically compatible.

[0036] The active constituents are used in a concentration sufficient toinhibit plant pathogen development of the targeted plant pathogen whenapplied to the cereal plant. As will be apparent to a skilled in theart, effective concentrations may vary depending upon such factors as:(a) the type of the plant or agricultural commodity; (b) thephysiological condition of the plant or agricultural commodity; (c) theconcentration of pathogens affecting the plant or agriculturalcommodity; (d) the type of disease injury on the plant or agriculturalcommodity; (e) weather conditions (e.g. temperature, humidity); and (f)the stage of plant disease. According to the invention, typicalconcentrations are those higher than 1×10² CFU/ml of carrier. Preferredconcentrations range from about 1×10⁴ to about 1×10⁹ CFU/ml, such as theconcentrations ranging from 1×10⁶ to 1×10⁸ CFU/ml. More preferredconcentrations are those of from about 37.5 to about 150 mg/g of drybacterial mass per of carrier (dry formulation) or per ml of carrier(liquid composition).

[0037] The compositions of the invention may be applied to the wheatplant or other cereals using conventional methods such as dusting,injecting, rubbing, rolling, dipping, spraying, or brushing, or anyother appropriate technique which does not injury the wheat plant orother cereals to be treated. Particularly preferred is the spray method.

[0038] The following specific examples are presented to moreparticularly illustrate the invention and should not be construed as alimitation thereon.

EXAMPLE 1

[0039] Effect of Bioprotectant Microorganisms of the Present Inventionon Radial Growth of Gibberella zeae in Vitro.

[0040] In paired treatments, the radial growth (cm) of Gibberella zeae,in the presence of the isolate ATCC PTA 3461., corresponding to Bacillusmegaterium (Embr. 9790) and the isolate ATCC PTA 3460, corresponding toPantoea agglomerans (Embr. 1494), was reduced substantially.

[0041] The antagonist activity of the isolated microorganisms wasdetermined by using the Antibiosis method as described in Luz (1990).Thousands of microorganisms were tested against Gibberella zeae. Eachisolate was transferred onto Petri dishes containing nutrient agar in acircular pattern by means of a small sterile glass funnel. After 48hours of incubation at 22° C. to 25° C., an agar disk containing a G.zeae colony was transferred into the center of the ring-shaped colony ofthe bioagent or, in the control treatment, onto an uninoculated mediaplate. The plates were incubated under fluorescent lights at 22° C. andunder a photoperiod of 12 hours, in a completely randomized design. Theradial growth of the pathogen was measured after three to five days, andthe result was 2.5 to 3.0 cm. Further measurements were made to permitthe assessment of the experiment. The data were calculated as %inhibition.

[0042] The data were subjected to analysis of variance and the meanscalculated by the Fisher test.

[0043] The pathogen growth reduction varied from 74% to 79% for B.megaterium and 25% to 38% for P. agglomerans. The results are shown inTable 2. In this table, the means within a column are significantlydifferent (at p=0.05) from each other if they are followed by differentletters, according to Fisher's least significant difference test. TABLE2 Effect of bioprotectants on radial growth of Gibberella zeae RADIAL %RE- GROWTH DUCTION TREATMENTS run 1 run 2 run 1 run 2 Nontreated 2.00 c3.5 c — — ATCC PTA 3461 0.42 a 0.9 a 79 74 (Bacillus megaterium-Embr.9790) ATCC PTA 3460 1.23 b 2.6 b 38 25 (Pantoea agglomerans-Embr. 1494)

EXAMPLE 2

[0044] Biocontrol of Wheat Scab (Fusarium Head Blight) Under GrowthChamber Conditions.

[0045] Cultivar and growth conditions—A susceptible wheat variety to beinfected by Fusarium was produced in EMBRAPA-Centro Nacional de Pesquisade Trigo (CNPT), Brazil. This cultivar was planted under growth chamberproviding adequate environmental conditions. Healthy-like plants(without disease symptoms) were grown and transferred to suitable potsand maintained in 12 hours-period dark at 20 ° C.

[0046] Pathogen preservation and inoculum production—A pure culture ofG. zeae isolate, obtained by EMBRAPA-Centro Nacional de Pesquisa deTrigo (CNPT), Brazil, onto a BDA containing streptomycin medium wasstored at 4° C. to avoid previous bacteria growth. F. graminearuminoculum was obtained from conidia which have been inoculated onto a BDAmedium. Sporogenesis was observed after 3-5 days from the inoculation.

[0047] Antagonists treatment—The isolates were assessed under growthchamber conditions at the stage of initial flowering. Ears wereuniformly sprayed with a suspension containing each isolate. The earswere treated with suspensions containing only one isolate and in aconcentration of approximately 10⁷ CFU/ml. There were nine replicatesper treatment and one blank (water without microorganisms).

[0048] Pathogen Inoculation and disease assessment—The pathogen wasinoculated 24 hours after antagonists treatment by spraying the earswith an aqueous suspension containing 10⁴ conidia of G. zeae/ml. Theinoculated plants were stored in a humid chamber for 48 hours. Thedisease assessment was made 15 days after pathogen inoculation. Diseaseseverity was assessed by calculating the percentage of the scabbyspikelets.

[0049] Biocontrol agents namely, Isolate ATCC PTA 3461 (Bacillusmegaterium (Embr. 9790)) and Isolate ATCC PTA 3460 (Pantoea agglomerans(Embr. 1494)) significantly reduced the percentage of scabby spikeletswhen tested in growth chambers either using fresh bacterial cells orfermented and dry preparations. The results are shown in Table 3. Inthis table, the means within a column are significantly different (atp=0.05) from each other if they are followed by different letters. TABLE3 Biochemical of wheat scab (Fusarium head blight) as measured bydecrease in disease severity under growth chamber conditions. DISEASESEVERITY (%) TREATMENTS run 1 run 2 run 3 run 4 Nontreated 54 b 86 b 62c 68 b ATCC PTA 3461 18 a 12 a 15 a 14 a (Bacillus megaterium (Embr.9790) (fresh cell)) ATCC PTA 3460 21 a 10 a 12 a 12 a (Pantoeaagglomerans (Embr. 1494) (fresh cell) ATCC PTA 3461 (Bacillus megaterium— — 20 b 24 b (Embr. 9790)) (wettable powder) ATCC PTA 3460 (Pantoeaagglomerans — — 22 b 18 b (Embr. 1494)) (wettable powder)

EXAMPLE 3

[0050] Biocontrol of Scab of Wheat Under Field Conditions.

[0051] Field experiment, plant treatment with the antagonists of theinvention—The experiment was carried out in an agricultural Brazilianarea which is used for scab of wheat microbiological control, wherenatural disease infection occurs. The wheat variety used in the test wasa Fusarium graminearum susceptible variety. Wheat plants with eachtreatment (concentration of 10⁷ CFU) were sown in plots of 12 rows, 3 mlong. The space between rows was 20 cm. Treated plots in each experimentwere arranged in a randomized block design.

[0052] Wettable powder compositions containing, individually, isolateATCC PTA 3461 (B. megaterium) and isolate ATCC PTA 3460 (Pantoeaaglomerans) of the present invention were also assessed.

[0053] Disease Severity and Yield Assessment—After 15 days from theapplication of the antagonists of the invention to wheat plants, thenatural disease severity was assessed. Yield was also calculated inkg/ha.

[0054] As shown in Table 4, Isolate ATCC PTA 3461 (Bacillus megaterium(Embr. 9790)) and Isolate ATCC PTA 3460 (Pantoea agglomerans (Embr.1494)) significantly diminished the disease incidence up to 50% andseverity up to 67% in the field and the yield increase varied from 809to 861 kg/ha (Table 4).

[0055] These results show that the biological control measure usingthese two bioagents play an important role in the management of scab onwheat. TABLE 4 Biocontrol of wheat scab (Fusarium Head Blight) asmeasured by decrease in incidence and severity of the disease, and yieldimpact under field conditions DISEASE YIELD TREATMENTS IncidenceSeverity (kg/ha) Nontreated 60 b 30 b 3328 b ATCC PTA 3461 (Bacillusmegaterium- 30 a 10 a 4189 a Embr. 9790) ATCC PTA 3460 (Pantoeaagglomer- 29 a 11 a 4137 a ans Embr. 1494)

1. A biocontrol composition comprising: a) a mixture of at least onemicroorganism which is an antagonist against plant pathogens and b) acarrier for said at least one microorganism, wherein said at least onemicroorganism is a bacteria selected from the group consisting ofPantoea agglomerans and Bacillus megaterium and said at least onemicroorganism is present in an amount effective for inhibiting plantpathogen development.
 2. The biocontrol composition according to claim1, wherein the carrier is selected from the group consisting of water,aqueous solutions, slurries, solids, and dry powders.
 3. The biocontrolcomposition according to claim 2, wherein the solid carrier is selectedfrom the group consisting of peat, wheat, bran, vermiculite, clay, talc,bentonite, diatomaceous earth, fuller's earth, and pasteurized soil. 4.The biocontrol composition according to claim 2, wherein the dry powdercarrier is a wettable powder.
 5. The biocontrol composition according toclaim 1, wherein said at least one microorganism is present in an amountof at least 1×10² CFU/ml of carrier.
 6. The biocontrol compositionaccording to claim 5, wherein said at least one microorganism is presentin an amount ranging from about 1×10⁴ to about 1×10⁹ CFU/ml of carrier.7. The biocontrol composition according to claim 1, wherein said atleast one microorganism is Pantoea agglomerans.
 8. The biocontrolcomposition according to claim 7, wherein said at least onemicroorganism is Pantoea agglomerans having the identifyingcharacteristics of deposit ATCC PTA 3460 (Embr. 1494).
 9. The biocontrolcomposition according to claim 1, wherein said at least onemicroorganism is Bacillus megaterium.
 10. The biocontrol compositionaccording to claim 9 wherein said at least one microorganism is Bacillusmegaterium having the identifying characteristics of deposit ATCC PTA3461 (Embr. 9790).
 11. The biocontrol composition according to claim 1,wherein the plant pathogens belong to the Fusarium species.
 12. Thebiocontrol composition according to claim 1, wherein said compositionfurther comprises an additive selected from the group consisting offertilizers, pesticides, preservatives, surfactants, wetting agents,buffering agents, coating agents, abrading agents, and mixtures thereof.13. A process for inhibiting the development of a plant pathogenicdisease caused by Fusarium species on a wheat plant or cereals, saidprocess comprising applying to said wheat plant or cereals a compositioncontaining a carrier and at least one microorganism which is anantagonist against plant pathogens and which is selected from the groupconsisting of Pantoea agglomerans and Bacillus megaterium, in an amounteffective to inhibit plant pathogen development on said wheat plant orcereals.
 14. The process according to claim 13, wherein the antagonistagainst plant pathogens is the bacterium Pantoea agglomerans.
 15. Theprocess according to claim 14, wherein said bacterium is Pantoeaagglomerans having the identifying characteristics of deposit ATCC PTA3460 (Embr. 1494).
 16. The process according to claim 13, wherein theantagonist against plant pathogens is the bacterium Bacillus megaterium.17. The process according to claim 16, wherein said bacterium isBacillus megaterium having the identifying characteristics of depositATCC PTA 3461 (Embr. 9790).
 18. The process according to claim 13,wherein said cereal is selected from the group consisting of wheat,barley, and corn.
 19. The process according to claim 13, wherein saidplant pathogenic disease caused by Fusarium species is Fusarium HeadBlight.
 20. The process according to claim 13, wherein said carrier isselected from the group consisting of aqueous solutions, slurries,solids, and dry powders.
 21. The process according to claim 13, whereinsaid composition further comprises an additive selected from the groupconsisting of fertilizers, pesticides, preservatives, surfactants,wetting agents, buffering agents, coating agents, abrading agents, andmixtures thereof.
 22. The process according to claim 13, wherein saidamount effective to inhibit plant pathogen development on said wheatplant or cereals is 37.5 to about 150 mg of dry bacterial mass per g orml of carrier.
 23. The process according to claim 13, wherein said stepof applying is by spraying.
 24. A process for increasing yield wheatplant or other cereals, said process comprising applying to the plant acomposition containing a carrier and at least one microorganism selectedfrom the group consisting of Pantoea agglomerans and Bacillusmegaterium, in an amount effective to enhance yield increase of saidplants.
 25. The process according to claim 24, wherein the bacterium isPantoea agglomerans.
 26. The process according to claim 25, wherein saidbacterium is Pantoea agglomerans having the identifying characteristicsof deposit ATCC PTA 3460 (Embr. 1494).
 27. The process according toclaim 24, wherein the bacterium is Bacillus megaterium.
 28. The processaccording to claim 27, wherein said bacterium is Bacillus megateriumhaving the identifying characteristics of deposit ATCC PTA 3461 (Embr.9790).
 29. The process according to claim 24, wherein said other cerealis selected from the group consisting of barley and corn.
 30. Theprocess according to claim 24, wherein said carrier is selected from thegroup consisting of aqueous solutions, slurries, solids, and drypowders.
 31. The process according to claim 24, wherein said compositionfurther comprises an additive selected from the group consisting offertilizers, pesticides, preservatives, surfactants, wetting agents,buffering agents, coating agents, abrading agents, and mixtures thereof.32. The process according to claim 24, wherein said step of applying isby spraying.
 33. Pantoea agglomerans having the identifyingcharacteristics of deposit ATCC PTA
 3460. 34. Bacillus megaterium havingthe identifying characteristics of deposit ATCC PTA 3461.